Microscope with a sheet of light

ABSTRACT

A family of microscopes with illumination systems directing a sheet of light having an approximately planar extension in an illumination axis of an illumination beam path and in a transverse axis orthogonally oriented to the illumination axis. The microscopes have detection devices used to detect light that is emitted by a sample region. The detection devices including a detection lens system disposed in the detection beam path and an optical detection element spaced from a front lens of the detection lens system and independently adjustable thereof. The optical detection element continuously varies the size of a detection image field and/or continuously displaces a focal plane of detection in the P-region.

PRIORITY CLAIM

The present application is a National Phase entry of PCT Application No.PCT/EP2010/063665, filed Sep. 17, 2010, which claims priority fromGerman Application Number 102009044983.3, filed Sep. 24, 2009, thedisclosures of which are hereby incorporated by reference herein intheir entirety.

FIELD OF THE INVENTION

The invention relates to a microscope comprising an illumination devicewhich produces a light sheet to illuminate a sample region, the saidsheet having an approximately planar extension in the direction of anillumination axis X of an illumination beam path and in the direction ofa transverse axis Y lying across the illumination axis X. The microscopefurther comprises a detection device used to detect light that isradiated by the sample region along a detection axis Z of a detectionbeam path, the illumination axis X and the detection axis Z as well asthe transverse axis Y and the detection axis Z being oriented relativeto each other at an angle unequal to zero. Preferably, the respectiveaxes are oriented approximately normal to each other.

BACKGROUND OF THE INVENTION

Such a microscope design comes under the category known as SPIMmicroscopes (SPIM—Selective Plane Illumination Microscopy). In contrastto confocal laser scanning microscopy (LSM), in which athree-dimensional sample is scanned point by point in individual planesat different depths and the image information obtained thereby issubsequently assembled to form a three-dimensional image of the sample,the SPIM technology is based on wide-field microscopy and permits theimaging of the sample on the basis of optical sections through variousplanes of the sample.

The advantages of the SPIM technology consist, among others, in thegreater speed at which the image information is detected, the reducedrisk of bleaching of biological samples, and a greater penetration depthof the focus into the sample.

The principle of SPIM technology is that fluorophores contained in thesample originally or added to it for contrasting are excited with laserlight, the laser radiation being shaped into a so-called light sheet.The light sheet is used to illuminate a selected plane in the depth ofthe sample in the sample region, and an imaging lens system is used toobtain an image of this sample plane in the form of an optical section.

First modern approaches to SPIM technology are described by A. H. Voieet al., Journal of Microscopy, Vol. 170 (3), pp. 229-236, 1993. Here,the fundamentals of modern SPIM technology are explained, in which acoherent light source is used to illuminate a sample, the light sheetbeing produced with the aid of a cylindrical lens. Arranged normal tothe propagation direction of the light sheet, which has a finitethickness, though, are detection means comprising an imaging lens systemand a camera.

In recent years, the technology was developed further, especially withregard to its application in fluorescence microscopy. For example, DE102 57 423 A1 and, based on it, WO2004/053558A1 describe methods inwhich a light-sheet-like illumination is produced due to a relativemovement between a line-shaped field of light and the sample to beexamined. The light-sheet-like illumination is formed by the field oflight being repeated in a temporal succession so as to be lined up sideby side due to the relative movement. In this way, though, shadows areformed within the sample plane to be examined, on account of parts ofthe sample that lie in the direction of illumination and are nottransparent to the illuminating light. Similar setups are also describedby Stelzer et al., Science (305) pp. 1007-1009 (2004), and Reynaud etal., HFSP Journal 2, pp. 266 (2008).

Instead of a purely static light sheet, for the generation of which acylindrical lens system is used, it is possible to produce aquasi-static light sheet by rapidly scanning the sample with arotationally symmetric light beam. The integration time of the camera onwhich the sample is imaged is chosen so that the scan is completedwithin the integration time. Such setups are described, e.g., by Kelleret al., Science (322), pp. 1765 (2008), and Keller et al., CurrentOpinion in Neurobiology 18, pp. 1-9 (2009).

All the setups and methods known in prior art, however, have more orless grave disadvantages, which restrict the use of the SPIM technologyin the commercial sphere, where it is important, among other things, toachieve a high user friendliness of the microscopes and, as a rule, ahigh throughput, with a great number of samples having to be examinedwithin a relatively short time. Essential disadvantages are describedbelow.

In most of the setups using SPIM technology that have been implementedso far, e.g., those according to DE 102 57 423 A1 and WO2004/053558A1,the mere variation of the image field size for detection—e.g., switchingfrom an image field size providing a good overview of the sample to adetail region—is rather a complex and time-consuming affair. It can onlybe implemented by a change of the detection objective. This affects thesample space unfavorably, which may have a particularly negative effectin case of a horizontal detection beam path. In the worst case, it alsoinvolves the removal and emptying of the sample chamber. After this,refocusing is necessary as a rule. Moreover, the sample is unnecessarilyheated or cooled.

An improvement is described by Becker et al., Journal of Biophotonics 1(1), pp. 36-42 (2008). Here, the detection beam path is arrangedvertically, so that a change of the image field size can be carried outwithout any substantial interaction with the sample chamber volume. Thedetection objective can be put into the sample chamber and taken outfrom above in a simple manner. Nevertheless, slight interactions withthe sample chamber and, thus, indirectly with the sample cannot beavoided.

Adaptation of the image field size is even simpler if zoom detectionobjectives are used. Such a setup is described, e.g., by Santi et al.,Biotechnics 46, pp. 287-294 (2009). Here, a commercial microscope, theOlympus MVX10, which has a zoom objective, is used for detection. This,too, is inserted into the sample chamber from above, which is, as arule, filled with an immersion liquid, so that, here again, there areslight interactions with the sample chamber when the zoom function ofthe objective is working or when the focus is adjusted, alone because ofthe motorized shifting of the lenses, which may cause vibrations thatmay transmit to the liquid in the sample chamber.

If the image field size for detection is changed, it is also desirableto adapt the illumination-side image field, i.e. to adapt the extensionof the light sheet along the transverse axis Y and the detection axis Z.In prior art, this adaptation has so far been implemented by the use ofinterchangeable diaphragms and/or beam expanders, as described, e.g., byKeller et al., Science 322, pp. 176 ff. (2008), and by Huisken et al.,Optics Letters 32 (17), pp. 2608-2610 (2007). The flare occurring incase of diaphragms causes light losses, whereas the use of beamexpanders reduces flexibility, since exchanging them is ratherlaborious.

While in the classical way, as described, e.g., in WO2004/053558A1, thelight sheet is produced via cylindrical lenses arranged in the beampath, the recent state of prior art, as described, e.g., in theabove-mentioned article by Keller et al., Science 322, pp. 176 ff.(2008), uses setups in which no static light sheet is produced butmerely a quasi-static light sheet, where the sample is rapidly scannedby a rotationally symmetric light beam. ‘Rapidly’ means that theintegration time of the spatially resolving array detector used as arule, e.g., a camera with CCD chip or CMOS chip, is chosen so that thelight beam scans the sample region corresponding to the quasi-staticlight sheet within this integration time. The integration time—which, inthe camera, e.g., corresponds to the shutter opening time—and thescanning frequency or scanning time of the light beam may, as a rule, beset independently of each other, so that the scanning time can beadapted to a fixed integration time. As scanning with a rotationallysymmetric light beam also produces a light sheet, at least in effect,this approach is also subsumed under the generation of a light sheet.

Both kinds of light sheet generation have advantages and disadvantages.With the use of cylindrical lenses, e.g., there is less of a load on thesample, because the intensity with which the sample is irradiated can beselected at a lower level while nevertheless the same dose is achievedas in case of scanning. Also, the use of cylindrical lenses is wellsuitable for recording image sequences in fast succession within veryshort times, since the speed is not limited by movable elements in theillumination beam path. In particular, a stroboscope-like illuminationcan be implemented very well with the use of cylindrical lenses. Inscanning, the swiveling scanning mirror used, as a rule, is apt to bethe speed-limiting element. If plain scanning is combined with angularscanning, i.e. illumination from different angles, in order to reducebanding as described, e.g., in DE 10 2007 015 063 A1, there is a riskthat beat artefacts will be produced if the scanners for light sheetangle scanning and position scanning are not matched, i.e. notsynchronized.

Advantages of light sheet generation by scanning are given by, amongother things, the fact that it permits a more homogeneous illuminationof the sample, so that quantitative image evaluations are possible aswell, which by the use of a cylindrical lens system can be achieved onlyapproximately by flaring through a diaphragm, which entails lightlosses. Moreover, a flexible choice of the maximum deflection of thescanner will permit the size of the image to be adapted with highflexibility. Scanning reduces the spatial coherence of the excitationlight, which also leads to a reduction of banding. Finally it ispossible, by special modulations of the light source, e.g. with an AOTF,to project grid patterns into the sample.

In other setups described in prior art, the sample is illuminated fromboth sides, from opposite directions along the illumination axis X. Inthe setup described by Santi et al., Biotechnics 46, pp. 287-294 (2009),the sample is illuminated simultaneously from both sides. For many kindsof samples, such as embryos of the fruit fly (Drosophila), such a setupis not of advantage, because in this way scattering and non-scatteringimage portions are combined in an unfavorable way. Huisken et al.,Optics Letters 32(17), pp. 2608-2610 (2007), and Becker et al., Journalof Biophotonics 1 (1), pp. 36-42 (2008), describe setups that illuminatethe sample sequentially, i.e. alternately from the two directions alongthe illumination axis X, which is more favorable for the samplementioned above. For switching back and forth between the twoillumination directions, a vibration-producing shutter or a rotatingmirror is used, so that the times required for switching are relativelylong.

Keller et al., in Science 322, pp. 1765 ff. (2008) and in CurrentOpinion in Neurobiology, 18, pp. 1-9 (2009), describe an SPIM setup inwhich the illumination and/or detection objective is mounted on a piezomotor, which permits focusing. Here, then, setting the focusing distanceis accomplished via a displacement of the entire objective. Inparticular, the distance of the front lens from the image plane is notmaintained, so that an interaction with the sample chamber is possible.This applies especially to horizontal detection beam paths with immerseddetection objectives: Here, the necessary movement of the objectiveentails tightness problems. On the other hand, a movable element in thesample space is disturbing in general, as the user may need space therefor diverse means for feeding to the sample chamber. The vibrationsoccurring during the movement of the objective may be unfavorablytransmitted to the sample, since the space between the objective and thesample is occupied by a liquid rather than by air.

If scanners are used for producing the light sheet, the imaging of thescanner into the pupil of the illumination objective is not optimal, asa rule, so that the plain position scanning is superposed by portions ofangular scanning.

Also known in prior art are setups in which the detection beam path issplit up into two branch beam paths; this is described, e.g., in the twopublications by Keller et al. mentioned above. For the beam splittingone uses beam splitters which transmit part of the light into one branchbeam path and reflect the other part of the light into the other branchbeam path. For this purpose one uses common dichroic filters having arelatively small thickness of less than 2 mm, which are arranged in adivergent part of the detection beam path. The advantage of such anarrangement is that in the direction of transmission there occur hardlyany artefacts caused by astigmatism. In the direction of the reflectedlight, however, image artefacts such as astigmatism or also defocusingdo occur, due to surface tensions at the dichroic filter, which can becaused, e.g., by the coating, or by improper installation. Another wayof splitting into two branch detection beam paths is described byHuisken et al. in Optics Letters 32, pp. 2608-2610 (2007). Here, thedichroic filter is located at infinity (related to the beam path), sothat, here again, the problems occurring in transmission are minimized.As far as the reflected branch beam path is concerned, though, theproblem of surface tensions may occur here, too, if conventionaldichroic filters are used.

SUMMARY OF THE INVENTION

A microscope comprising an illumination device which produces a lightsheet to illuminate a sample region, the said sheet having anapproximately planar extension in the direction of an illumination axisX of an illumination beam path and in the direction of a transverse axisY lying across the illumination axis X. The microscope further comprisesa detection device used to detect light that is radiated by the sampleregion along a detection axis Z of a detection beam path, theillumination axis X and the detection axis Z as well as the transverseaxis Y and the detection axis Z being oriented relative to each other atan angle unequal to zero. Preferably, the respective axes are orientedapproximately normal to each other.

In an embodiment of the invention, the detection device is provided withan optical detection element that is arranged spatially separate from afront lens of the detection objective and that can be adjustedindependently of this front lens, the said optical detection elementbeing used to continuously vary the size of a detection image fieldand/or to continuously displace a detection focal plane in the sampleregion. Front lens in this context means the lens, or a correspondingcemented component, that is located first in the beam path, i.e.,closest to the sample region.

An advantage of embodiments of the invention is that the size of thedetection image field can be continuously varied by means of the opticaldetection element; secondly, the detection element can be used tocontinuously displace a detection focal plane in the sample region. Thedetection element may be designed in such a way that it performs onlyone of these two tasks, or can be used to perform both settings eitheralternately or simultaneously. The detection element may be an integralpart of the detection objective, say, in the form of two or more lenscomponents movable relative to each other, with the front lens remainingstationary during the movement of these lens components. Alternatively,the detection element may be a separate component in the beam path,arranged at some distance from the detection objective.

In an embodiment, the optical detection element may be designed as adetection zoom element. For example, the detection zoom element maycomprise two movable lens components and a fixed lens component inbetween. Other configurations of the detection element with but a singlemovable lens component, or with the fixed lens component locatedelsewhere, or with more than two movable lens components are feasible aswell. The use of a detection zoom element enables simple switchingbetween an overview image and a detail image, and efficient locating ofa sample region of interest. For the automated generation of tables ofcontrol element positions, one can use an intermediate image sample suchas, e.g., a transmission grid pattern inserted in an intermediate imageplane of the detection beam path. Alternatively, a calibration objectivecan be used. By suitable shifting of the detection zoom element it isalso possible to compensate various chromatic aberrations of thedetection device; different tables can be generated for differentemission wavelengths and zoom positions. To compensate the saidaberrations, the focal plane in the sample region is shiftedcontinuously by means of the detection zoom element, going by the tablesif necessary. Because of the spatial separation between the front lensof the detection objective and the detection zoom element, the detectionobjective itself, or, in particular, the front lens of the detectionobjective need not be moved; by means of the detection zoom element,which is arranged at a permanent location in the detection beam path,so-called internal focusing of the detection beam path can be performed.This offers advantages both in handling the sample and in recordingimage stacks along the detection direction.

In another embodiment, the detection direction can be provided with atube lens unit arranged spatially separate from the detection elementand from the detection objective, by means of which tube lens unit thedetection focal plane can be shifted in the sample region. In this case,the detection element, particularly the detection zoom element, is usedonly for changing the image field size, whereas the adjustment of thefocal plane is done, e.g., by shifting a suitable tube lens or a tubelens element of the tube lens unit. The combination of both ways, i.e.,with the detection focal plane being adjustable with both the tube lensunit and the detection zoom element, is another possible embodiment.

In another embodiment of the invention, the illumination device in theillumination beam path is provided with at least one illuminationobjective and an optical illumination element that is arranged spatiallyseparate from a front lens of the illumination objective and isadjustable independently of it, by means of which optical illuminationelement the extension of the light sheet in the direction of thedetection axis Z is continuously variable and/or by means of which anillumination focal plane in the sample region can be continuouslyshifted. With the illumination element, then, it is possible tocontinuously adapt the illumination-side image field size ormagnification, or also to define the focus. The illumination element maybe an integral part of the illumination objective—say, in the form oftwo or more lens components that are movable relative to each other,with the front lens remaining stationary during the movement of theselens components. Alternatively, the illumination element may be aseparate component in the beam path arranged at some distance from theillumination objective.

This is possible in a particularly simple manner if the illuminationelement is designed as a illumination zoom element with preferably atleast three lens components that can be shifted independently of eachother. If the detection device is provided with a detection zoomelement, the illumination zoom element can be used to optimally andcontinuously match the numerical aperture of the illumination to thesize of the detection image field. In this way, the frequency ofchanging the illumination objectives is reduced, and sample stress isalways kept at the lowest possible level. The ratio betweendetection-side image field size and illumination-side image field sizecan be set in such a way that a ratio of the light sheet thicknesses of1 at the center to 2 on the margin of the field of view is set and keptconstant when the image field size is varied.

The use of three shiftable elements is advantageous in so far as, inaddition to the correct magnification, the correct imaging of pupil andintermediate image can be guaranteed. Altogether, adjustments can bemade in three degrees of freedom by means of the three separatelyshiftable elements. Where adjustments are needed in fewer degrees offreedom, correspondingly fewer shiftable lens elements are required inthe detection zoom element.

For adapting the illumination-side image field size to the size of thedetection image field it is of advantage if the illumination zoomelement and the detection zoom element are coupled via a controlcircuitry for setting the illumination zoom elements as a function ofthe detection image size given by the detection zoom element and also,of course, by the static magnification of the other detection opticalcomponents. In this case, if the size of the detection image field isvaried, e.g. manually by the observer, the illumination-side image fieldsize is automatically adapted to this variation. The control circuitrymay, of course, also be conceived for detection and/or illuminationelements, which are part of the respective detection or illuminationobjective.

The invention further provides a microscope wherein the illuminationdevice additionally comprises first means for light sheet generation,which, in turn, comprise means for generating a rotationally symmetriclight beam and scanning means for light-sheet-like scanning of thesample region along the transverse axis in a specified time interval,with the illumination device further comprising second means for lightsheet generation, which in turn comprise a first astigmatically actingoptical element with at least one astigmatic lens for generating astatic light sheet, and in which microscope there are further providedselecting means with which, for generating the light sheet, either thefirst or the second means for light sheet generation or both togethercan be selected. As an astigmatically acting lens, one can use, e.g., acylindrical lens, but some other astigmatically acting lens such as aPowell lens may be used as an equivalent.

Whereas the first means for light sheet generation generate aquasi-static light sheet by means of a fast scanning mirror, the secondmeans for light sheet generation generate a static light sheet. In thisway, the advantages of scanning light sheet generation can be combinedwith those of light sheet generation by means of a cylindrical lenssystem. For expediency, the scanning means comprise a rapidly switchablescanning mirror and a scanning objective. One can, selectively, use oneor the other method of light sheet generation; thus it is possible,e.g., to leave the scanning mirror in its zero position and, by means ofthe cylindrical optical element, to generate a static light sheet, whichilluminates the sample in the manner of a stroboscope, for which thescanning mirror is too slow. In addition, angle scanning means may beprovided, by means of which the angle between the light sheet and theillumination axis can be varied. The angle scanning means, too, maycomprise a rapidly switchable angle scanning mirror. This may be, forexample, a resonance scanner of the microelectromechanical type. As arule, the angle scanning mirror operates at a frequency of 10 kHz,whereas the scanning mirror for generating the light sheet operates at afrequency approximately between 1 kHz and 2 kHz.

The angle scanning mirror may be arranged conjugated to the illuminationfocal plane if the second means for light sheet generation have beenselected, i.e., if, for example, the cylindrical optical element is inthe beam path. By means of the angle scanning mirror, the sample can beilluminated from different angles, which can be used to reduce banding.

In addition to the fast scanning mirror for light sheet generation, andin its immediate vicinity, another fast scanning mirror may be placed inthe beam path, with which, e.g., the light sheet can be shifted in thedirection of the detection axis for adjusting purposes, or the light canbe deflected into another illumination beam path; the latter action,however, can also be performed by means of a separate switching mirror.

In addition, then, a second astigmatically acting optical element, whichalso may be configured as a cylindrical optical element, may be arrangedin the illumination beam path. This serves to correctly image one of thetwo scanning mirrors onto a pupil plane, while the other scanning mirroris correctly imaged without the action of the cylindrical opticalelement. Essentially, this second element is a corrective optical systemfor the correct imaging of the two scanning mirrors mentioned.

Embodiments of the invention further provides a microscope in which theillumination device additionally comprises means for deflectingillumination light in another illumination beam path and for generatinganother light sheet, which is of approximately planar extension in thedirection of the illumination axis and in the direction of thetransverse axis, with the illumination beam path and the otherillumination beam path are constructed of essentially identical opticalelements and have identical optical path lengths, and with the lightsheet and the other light sheet being aligned relative to one another insuch a way that they illuminate the sample region from oppositedirections on the same illumination axis, the illumination devicefurther comprising switching means for switching the illumination lightbetween the illumination beam path and the other illumination beam path,and the detection device comprising a detection objective for imaginglight radiated from the sample region light onto an array detector forlocus-dependent detection of that light, with the switching meanscomprising a rapidly switchable switching element having a switchinginterval of less than 10 ms, with a specified integration time of thearray detector and the switching interval of the switching elementsbeing tuned to each other in such a way that, during the integrationtime, the sample region is illuminated at least once from eitherdirection on the illumination axis.

Therefore, the integration times for the array detector, i.e., the timewithin which, e.g., the shutter of a camera is open and measurement dataare collected, are minimized, or the signal-to-noise ratio may beinfluenced adversely. It is of advantage, therefore, to use agalvanometer-driven mirror having a switching interval of less than 10ms.

Instead of a switching mirror one may use other switching elements, suchas acousto-optical or electro-optical switching elements, which mayoperate on a reflective or, equivalently, on a transmissive basis.

What is important is that, compared to the integration time of the arraydetector (which does not exceed 20 ms, as a rule, as otherwisevibrations could have an adverse effect), the switching interval shouldbe short enough to permit the sample region to be illuminated at leastonce from either direction within the integration time. Multiple scansfrom either direction are feasible as well if the switching interval issmall enough, e.g., less than 5 ms.

In this way, rapidly switchable two-beam illumination can be achieved,which can be used for sequential alternating illumination of the samplefrom two sides and for quasi-simultaneous illumination of the samplesfrom two sides, depending on the nature of the sample. The term“quasi-simultaneously” is used because the illumination directionchanges during the integration time of the camera, and—in case the lightsheet is generated in a scanning manner, with the sample being scannedby a rotationally symmetric light beam—the light beam scans the sampleat least twice, but from different directions. With the short switchingintervals mentioned, the sample does not change its position, as a rule,so that in the image, in effect, the sample appears as illuminated fromboth sides.

In effect, then, the sample in the sample region may be illuminatedsimultaneously from opposite directions along the illumination axis, inthat, during the integration time of the array detector, theillumination light is switched at least once from one illumination beampath to the other.

The switching mirror may be favorably arranged in a plane conjugated tothe illumination pupil. This has the advantage that, for switching over,the positional offset is utilized that is created by the deflection ofthe switching mirror and by using the scanning objective. Alternativesetups in which the angular offset is used directly for switching arefeasible as well.

The illumination device may also comprise means for light sheetgeneration, which in turn comprise means for generating a rotationallysymmetric light beam and scanning means for light-sheet-like scanning ofthe sample region along the transverse axis, with the scanning meanscomprising, e.g., a rapidly switchable scanning mirror and a scanningobjective. Optionally, angle scanning means may be provided, by means ofwhich an angle between the light sheet and the illumination axis isvariable. These angle scanning means may comprise, for example, arapidly switchable angle scanning mirror for the reduction of banding.

In addition to the fast scanning mirror for light sheet generation, andin its immediate vicinity, another fast scanning mirror may be placed inthe beam path, with which, e.g., the light sheet can be shifted in thedirection of the detection axis for adjusting purposes. This can also beperformed by means of the switching mirror.

With the setup suitably configured, the other fast scanning mirror orswitching mirror may, by a slight offset, also be used for adjusting thelight sheet in the detection direction.

Herein it can be of advantage that the illumination device comprises anastigmatically acting optical element, preferably a cylindrical opticalelement, which is arranged in the one illumination beam path or in theother illumination beam path and serves to correctly image the scanningmirror, the switching mirror or the other scanning mirror onto a pupilplane. Whereas one of the two mirrors is correctly imaged onto the pupilplane, imaging of the other mirror without such an additionalastigmatically acting element would not be correct; this element, then,is an optical correction system for the correct imaging of both mirrors.

An interesting application results if the array detector has areas ofpixels which can be read out separately and for which differentintegration times can be specified, as it is the case, e.g., with modernarray detectors on CMOS basis. Here, the switching interval of theswitching element can additionally be synchronized with these differingintegration times.

Embodiments of the invention further provide a microscope of in whichthe detection device additionally comprises in the detection beam path adetection objective and splitting means for splitting the detection beampath into two branch beam paths, with a spatially resolving arraydetector arranged in each of the branch beam paths, on which arraydetector the light to be detected is imaged, and with the splittingmeans in turn comprising at least one dichroic beam splitter, thedichroic beam splitter being arranged in the beam path in thenear-infinity space relative to the spatially resolving array detectorsand having a thickness of at least 3 mm, preferably upwards of at least4 mm, and optical imaging elements for the imaging of the light to bedetected onto the respective array detector being arranged in each ofthe two branch beam paths, and at least one wobble plate being arrangedin at least one of the two branch beam paths, for generating a beamoffset along two mutually orthogonal directions transversely to thedetection axis. In this way, superpositions of the measurement data readout of the two array detectors can be achieved automatically. Inparticular, adaptation to different camera types possibly employed byend users can be accomplished without problems. If only one wobble plateis used, it must be possible to set it to the two orthogonal directions.With equivalent effect, one can use two wobble plates. One of them isthen used to adjust the beam offset in one direction, while the otherplate is used to adjust the beam offset in the direction orthogonal toit.

In embodiments of the invention a transmission pattern, e.g., atransmission grid pattern, may be placed in an intermediate image planeof the detection beam path, this transmission pattern being selectableand serving to aligning the images with each other. Alternatively, acalibration objective can be used in the position of the detectionobjective. Once the images have been aligned, the transmission gridpattern or the calibration objective can be removed again from the beampath. Superposition of the images can then be accomplishedautomatically. Setting the offset may be performed by means of thewobble plates, the wobble plates may be arranged in one of the twobranch beam paths.

The optical imaging elements in the respective branch beam paths can bearranged so as to be shiftable, so that focusing onto the respectivearray detector is possible. This is of advantage particularly forcompensating longitudinal chromatic aberrations, e.g., if measurementsare carried out on samples marked with two different dyes and thespectral emission ranges or emission wavelengths are to be detected inthe two branch beam paths. It may be sufficient if at least one of theoptical imaging elements can be shifted, if focusing can be performed byother means provided.

As an addition or an alternative, each of the two branch beam paths maybe provided with a wedge assembly consisting of two optical wedgesprotruding into the beam path and shiftable relative to each othertransversely to the beam direction, this wedge assembly being arrangedbetween the optical imaging element and the array detector, i.e. in thedivergent part of the beam path, so that focusing on the respectivearray detector can be achieved by shifting the wedges relative to eachother.

Instead of arranging the beam splitter in the near-infinity space of thebeam path, the dichroic beam splitter may also be arranged in thedivergent part of the beam path between the array detectors and anoptical imaging element for imaging the light to be detected onto thearray detectors. In the branch beam path that captures the lighttransmitted by the beam splitter, then, a glass plate of identicalthickness as the beam splitter may be arranged between array detectorand beam splitter, the glass plate and the beam splitter enclosing anangle of approximately 90°. In this way, any astigmatism caused by theuse of the beam splitter in the transmission branch beam path iscorrected.

In an embodiment, this glass plate is designed as a wobble plate;therefore, it can be used also for generating a beam offset transverselyto the beam direction, so that, here again, automatic superposition ofthe measurement data read out of the array detectors can beaccomplished.

For changing the focus position in a simple embodiment, the opticalimaging element can be arranged so as to be shiftable, so that it can beused to focus on the array detectors. If two different wavelengths aremeasured, it can be assumed that, for at least one of the twowavelengths, focusing is not exact. To avoid this, a wedge assemblyconsisting of two optical wedges that protrude into the beam path andcan be shifted transversely to the beam direction is arranged in atleast one of the two branch beam paths between the beam splitter and thearray detector, thus allowing focusing on the respective array detector.In this case, first the beam path not containing the wedge assembly,e.g., the transmission branch beam path, is focused by means of shiftingthe optical imaging element. This is done for one of the two wavelengthsto be measured. Then the two wedges in the other beam path, e.g., thereflection branch beam path, are shifted relative to each other for fineadjustment for the other wavelength. As an alternative, the opticalimaging element may be arranged in a fixed position, and wedgeassemblies may be arranged accordingly in each of the branch beam paths.

It is understood that the features mentioned before and those to beexplained below are applicable not only in the combinations stated butalso in other combinations or as stand-alone features without leavingthe scope of the present invention.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a detection beam path for an SPIM microscope,

FIG. 2 shows three different settings of the illumination beam path foran SPIM microscope,

FIG. 3 shows details for the settings shown in FIG. 2,

FIG. 4 shows another illumination beam path,

FIG. 5 shows an illumination beam path for illumination from two sides,

FIG. 6 shows the effects the illumination from two sides has on asample,

FIG. 7 shows a detail of a detection beam path with two channels, and

FIG. 8 shows a detail of another detection beam path with two channels.

DETAILED DESCRIPTION

FIG. 1 shows a detection beam path of a microscope operating by theprinciple of SPIM technology. Pertaining to it, though not shown in FIG.1, is an illumination device of the microscope, with which a light sheetfor illuminating a sample region P is generated. In the direction of anillumination axis X of an illumination beam path and in the direction ofa transverse axis Y lying across the illumination axis X, the lightsheet has an approximately planar form. Shown along the detection beampath are elements of a detection device 1 used to detect light that isradiated by the sample region P along a detection axis Z. Theillumination axis X and the detection axis Z are approximatelyperpendicular to each other, as are the transverse axis Y and thedetection axis Z.

The detection device 1 comprises a detection objective 2 arranged in thedetection beam path. Other essential elements in the detection beam path1 are a tube lens unit 3 and a spatially resolving array detector 4,which may be designed, e.g., as a CCD chip or as a CMOS chip of asuitable camera. The light is imaged onto this array detector 4 by meansof an optical imaging element 5.

An element of the detection device 1 is an optical detection element,which is arranged so as to be separated from the front lens of thedetection objective 2 and can be adjusted independently of this frontlens. By means of the optical detection element, on the one hand, thesize of a detection image field is continuously variable, and on theother hand, the detection element can be used to continuously shift adetection focal plane in the sample region P. The detection element canbe designed so as either to serve only one of the two tasks or bothtasks alternatively, or so that both settings can be carried outsimultaneously. The detection element may be an integral part of thedetection objective 2, e.g., in the form of two or more lens componentsthat are movable relative to each other, whereas the front lens remainsstationary during the movement of these lens components. Alternatively,the detection element may be arranged as a separate component in thebeam path at a distance from the detection objective 2. During anadjustment of the detection element, i.e., a shifting of one or severalof its lens components along the beam path, the front lens remainsstationary.

The optical detection element as a separate component may be designed,e.g., as shown in FIG. 1, as a detection zoom element 6 with two movablelens components 6.1 and 6.2 and a fixed lens component 6.3 between them.The use of a detection zoom element 6 enables easy switching between anoverview image and a detail image, or efficient locating of the sampledetails of interest. Furthermore, by means of the detection zoom element6, the recording of image stacks along the detection direction Z atdifferent sample angles—so-called multiview image stacks—is possible inan easy way. The detection zoom element 6 can be adjusted completely bymotor drive. For the automatic compilation of tables of control elementpositions, one can use an intermediate image sample such as atransmission pattern, e.g., a transmission grid pattern 7, which ismoved into an intermediate image plane of the detection beam path. InFIG. 1, this transmission grid pattern 7 is shown in the moved-in state;however, it can also be moved out and is not needed for detection onceall parameters have been ascertained. Alternatively, a calibrationobjective (not shown) can be used in the beam path instead of thedetection objective 2.

The setup shown can also facilitate the recording of so-calledmultitrack micrographs at different wavelengths of light, as one canmake use of the fact that different tables for position settings can becompiled for different emission wavelengths. By adjusting the detectionzoom element 6 accordingly, one can thus compensate longitudinalchromatic aberrations of the detection device 1.

It is advantageous for this purpose to continuously shift the focalplane in the sample region P by means of the detection zoom element 6.Since the detection zoom element 6 is arranged separately from thedetection objective 2, the masses to be moved are very small, as thedetection objective 2 itself need not be moved. By means of thedetection zoom element 6, which is arranged in a stationary position inthe detection beam path, one can effect what is known as internalfocusing of the detection beam path. Because of the smaller masses to bemoved, image stacks along the detection direction Z, which requirerepeated focus adjustment, can be recorded at higher accuracies andspeeds.

Moreover, water sealing of the sample chamber is facilitated, since thedetection objective 2, or its front lens, does not move relative to thesample chamber, in which the sample is placed and whose sample region Pis illuminated. Interactions with the sample by vibrations are thusavoided, and no additional forces act on the sample. Moreover, it ispossible to specify the detection focal plane as a function of thetemperature of the liquid with which the sample chamber is filled, forwhich purpose the temperature of this liquid is measured and a focusposition is adjusted accordingly. If the temperature changes, thischange is signaled to the detection element via an evaluation unit and acontrol unit, and the detection focal plane is adjusted accordingly. Inthis way, a variation of the refractive index of the liquid with whichthe sample chamber is filled can be compensated. Frequently, water isused as a liquid or immersion medium. Refractive index data may bestored for different liquids; if the user specifies which immersionmedium is used, correct adjustment can then be carried outautomatically. This kind of temperature-dependent variation of thedetection focal plane can be used, for example, in so-called heat shockexperiments, in which the sample or the immersion liquid is subjected togreat temperature changes within a very short time.

In the example shown, the detection zoom element 6 is designed to havetwo movable lens components 6.1, 6.2, between which there is a fixedlens component 6.3; however, other designs with more movable lenscomponents or with only one movable lens component are possible. Forexample, it may quite well be provided not to use the detection zoomelement 6 for internal focusing, i.e. the continuous shifting of thedetection focal plane. In this case, one can use, e.g., a tube lens 8 ofthe tube lens unit 3 for shifting the detection focal plane in thesample region, i.e. for internal focusing. This tube lens 8, then, isarranged so as to be movable along the beam path, as shown symbolicallyby the double-headed arrow. The tube lens unit 3 is also arrangedseparately from the detection objective 2, and likewise so from thedetection element or detection zoom element 6.

The detection device 1 features several further optical elements, ofwhich some that are more essential are described below. To achieve acompact design, the beam path is directed from the sample region P tothe spatially resolving array detector 4 via deflecting mirrors 9 and10. By means of the beam coupler 11, an additional beam path canoptionally be coupled in, which can be used for reflected-lightillumination. Moreover, a dichroic beam splitter 13 may be arranged inthe beam path between the zoom element 6 and the imaging element 5 inthe near-infinity space 12 of the beam path, i.e., in a range in whichthe beam is collimated as much as possible but not necessarilycompletely, the said dichroic beam splitter 13 serving as a splittingmeans for splitting the detection beam path into two branch beam paths14 and 15, with a spatially resolving array detector on which the lightto be detected can be imaged being arranged in each of the branch beampaths 14 and 15. FIG. 1 shows only the optical elements for the branchbeam path 14; the branch beam path 15 may be of identical design.

The dichroic beam splitter has a thickness of more than 3 mm, so thatproblems such as imaging aberrations due to the occurrence of surfacetensions as known in prior art can be avoided. In each of the two branchbeam paths 14 and 15, optical imaging elements for the imaging of thelight to be detected onto the respective array detector are arranged. Inat least one of the two branch beam paths 14 or 15, wobble plates 16 and17 may optionally be arranged, with which an offset can be adjusted inthe two orthogonal directions transversely to the detection axis Z. Inthis way, an automatic superposition of the measurement data read outfrom the two array detectors can be accomplished. This is meaningful,e.g., if images for two different emission wavelengths are to berecorded separately and then superposed. For this purpose, FIG. 1 alsoshows an optional emission filter 18 in the branch beam path 14, withwhich a wavelength selection can be carried out. A correspondingemission filter 18, which selects another wavelength, may be arranged inthe other branch beam path 15. In addition, for the correct adjustmentof image superposition, an intermediate image sample may be used forcalibration, in such a way that the above-mentioned transmission gridpattern 7 is used in an intermediate image plane of the detection beampath, or that a calibration objective (not shown) is used instead of thedetection objective 2.

Furthermore, the optical imaging elements in respective branch beampaths may also be arranged so as to be shiftable, so that it is possibleto focus on either respective array detector.

FIG. 2 shows an illumination device 19, with which a light sheet toilluminate a sample region P is generated, which light sheet is ofapproximately planar form in the direction of an illumination axis X ofan illumination beam path and in the direction of a transverse axis Ylying across the illumination axis X. For the sake of clarity, adetection device 1 as shown as an example in FIG. 1 has been left outhere, but both can readily be combined with each other, with mutualalignment being accomplished in accordance with the coordinate systemsshown in FIGS. 1 and 2.

The illumination device 19 illustrated in FIG. 2 is shown in threedifferent configurations A, B and C. The illumination device 19comprises, in the illumination beam path, at least one illuminationobjective 20 and an illumination element that is arranged at a distancefrom a front lens of the illumination objective 20 and can be adjustedindependently of this front lens, by means of which illumination elementthe extension of the light sheet in the direction of the detection axisZ is continuously variable and/or by means of which an illuminationfocal plane in the sample region P can be shifted continuously. Theillumination element may be an integral part of the illuminationobjective 20, e.g., in the form of two or more lens components that aremovable relative to each other, whereas the front lens remainsstationary during the movement of these lens components. Alternatively,the illumination element may be arranged as a separate component in thebeam path at a distance from the illumination objective 20. During anadjustment of the illumination element, i.e., a shifting of one orseveral of its lens components along the beam path, the front lensremains stationary.

The illumination element can thus be used to continuously adapt thelight sheet to the image field size or to set the focus, respectively.This is possible in a simple way if, e.g., the illumination element isdesigned as an illumination zoom element 21. This can then be used,particularly if the detection device 1 is also provided with a detectionzoom element 6, to optimally and continuously match the numericalaperture of the illumination with the size of the detection image field.Compared to other solutions such as interchangeable telescopes,adaptation of the numerical aperture by means of the illumination zoomelement 21 can be done continuously. Compared to other solutions such asdiaphragms, no excitation light will be lost, so that, in the end, theillumination source required—as a rule a laser—may have a lower output.In this way, the load on the sample is reduced, higher power densitiescan be achieved, and the illumination objectives 20 need to be changedless frequently. The ratio between the size of the detection image fieldand that of the illumination image field can be adjusted in such a waythat a ratio of the light sheet thicknesses of 1 in the center to 2 onthe margin of the field of view can be set and kept constant when theimage field size is varied.

If it is possible in addition, by means of the illumination element, tocontinuously shift the illumination focal plane in the sample regionP—an adjustment that is also accomplished by internal focusing similaras in case of the detection zoom element 6, with one or several lenselements of the illumination zoom element 21 being shifted relative toeach other—, the waist of the light sheet can also be shiftedcontinuously across the field of view in the direction of theillumination axis X, a process called waist scanning. In this process,maximum resolution in the detection direction Z can be achieved all overthe field of view.

In combination with illumination from two sides along the illuminationaxis X, internal focusing permits the waists to be positioned to approx.¼ and approx. ¾ of the field of view. In this way, variations of thethickness of the light sheet can be kept distinctly smaller than in caseof illumination from one side only. Finally, in analogy totemperature-dependent focusing as described in connection with thedetection zoom element 6, compensation of the position of the lightsheet waist as a function of the temperature of the immersion medium orof differing optical thicknesses of the medium used for mounting thesample is possible.

For adapting the illumination-side image field size or the extension ofthe light sheet to the size of the detection image field, it is ofadvantage to couple the illumination zoom element 21 and the detectionzoom element 6 via a control circuitry for setting the illumination zoomelement 21 as a function of the size of the detection image field givenby the detection zoom element 6. In this case, if the size of thedetection image field is changed, e.g., manually by an observer, theillumination-side image field size is automatically adapted to thischange. The observer is thus spared illumination-side adaptation of theillumination zoom element 21, as this is accomplished by the controlcircuitry, so that the numerical aperture for illumination is always setat an optimum.

The illumination zoom element 21 shown in FIG. 2 is provided with atleast three independently shiftable lens components 21.1, 21.2 and 21.3and a fixed lens component 21.4. The beam paths A, B and C show threedifferent positions of the illumination zoom element 21. The use ofthree shiftable elements is necessary in this case in order to ensurenot only the correct magnification but also the correct imaging of pupiland intermediate image; altogether, then, three degrees of freedom canbe set. Where fewer degrees of freedom are relevant, fewer shiftableelements will suffice.

The effect of the illumination zoom element 21 is shown in some moredetail in FIG. 3 for the three different configurations A, B and C. Theillumination zoom element 21 reveals its effect both in the planespanned by the illumination axis X and the transverse axis Y, as shownin FIG. 3 a, and in the plane spanned by the illumination axis X and thedetection axis Z, as shown in FIG. 3 b. In FIG. 3 b one can see how, byadaptation of the illumination aperture, the light sheet thickness fromposition A to position B to position C of the illumination zoom element21 is rendered increasingly thinner. In FIG. 3 a, the adaptationresulting from the setting of the illumination zoom element 21 is shownin the X-Y plane.

To further increase the flexibility regarding the selection of theillumination aperture, the illumination device 19 may also comprise atelescope changer 22, which accordingly broadens the range of aperturesachievable. As an alternative or an addition, the illumination objective20 can be changed to generate a similar effect.

The illumination device 19 shown in FIG. 2 furthermore comprises firstmeans for light sheet generation, which in turn comprise means forgenerating a rotationally symmetric light beam and scanning means forlight-sheet-like scanning of the sample region along the transverse axisY in a specified time interval. Whereas the means for generating therotationally symmetric light beam are not shown, the scanning means ofthe setup shown in FIG. 2 comprise a scanning mirror 23, with which therotationally symmetric light beam is guided across the sample, at such aspeed that the scanner sweeps the sample at least once, but preferablyseveral times, and in any case completely, during the integration time,i.e. the time that passes before the measurement data are read out fromthe detector; in other words, the integration time must not end as longas the light beam still sweeps the sample. Integration time, scanningtime and scanning runs are tuned to each other, so that the specifiedtime interval essentially equals the integration time. The effect of thescanning mirror 23 is not shown in FIG. 2 and FIG. 3, though.

The illumination light leaving the scanning mirror 23 is imaged into thesample region P via a scanning objective 24, a tube lens element 25 andthe illumination objective 20. To make the setup as compact as possible,various mirrors 26 are used for beam deflection.

In addition to the scanning mirror 23, the illumination device 19 isoptionally provided with second means for light sheet generation, whichcomprise a first astigmatically acting optical element—in the exampleshown this is a first cylindrical optical element 27 with at least oneastigmatic lens, here a cylindrical lens—for generating a static lightsheet. Also provided (not shown) are selecting means, with which, forgenerating the light sheet, either the first or the second means forlight sheet generation or both together can be selected. This means thateither the scanning mirror 23 or the cylindrical optical element 27 orboth together are used. The cylindrical optical element 27 may bedesigned, e.g., in such a way that it can be moved into or out of thebeam path laterally. In this way, the advantages of both methods forlight sheet generation, each described above, can be combined. Insteadof a cylindrical lens, the first astigmatically acting optical elementmay also comprise a Powell lens or other astigmatically acting lenses.

In particular, the general coverage of the image field in the sampleregion P is linked to the cylindrical optical element 27, which, in thesetup shown here, is used simultaneously with the scanning mirror 23.The cylindrical optical element 27, though, cannot cover the full imagefield in every position of the illumination zoom element 21.

In FIG. 2, the illumination device 19 further comprises optional anglescanning means. In the embodiment shown in FIG. 2, the angle scanningmeans comprise a rapidly switchable angle scanning mirror 28. By meansof the angle scanning mirror 28, the angle between the light sheet andthe illumination axis X is variable. As an angle scanning mirror 28, onecan use, e.g., a resonance or polygon scanner, including such of amicroelectromechanical type. In the case of the first cylindricaloptical element 27 being used, the angle scanning mirror 28 is arrangedso as to be conjugate with the illumination focal plane. By means of theangle scanning mirror 28, the sample can be illuminated from differentangles, which can be used to reduce banding. In FIG. 2, the action ofthe angle scanning mirror 28 is illustrated for configuration C by thetwo beam paths 29 and 30. Whereas in beam path 29 the angle scanningmirror is in zero position, i.e., not deflected, beam path 30 applies incase of a deflection of the angle scanning mirror 28 different fromzero. This is shown again for all configurations A, B, C in FIG. 3 a.The angle scanning mirror 28 operates, as a rule, at a frequency of 10kHz, whereas the scanning mirror 23 for generating the light sheetoperates at a frequency approximately between 1 kHz and 2 kHz.

The use of the cylindrical optical element 27 is of advantage alsobecause, in case of simultaneous use of the angle scanning mirror 28,any beat artefacts that may occur due to asynchronous triggering of theangle scanning mirror 28 and the rapidly switchable scanning mirror 23are, in part, made indistinct by the cylindrical optical element 27placed in the beam path. As the use of the cylindrical optical element27 parallels the light beam, the intensity of illumination is reducedcompared to the sole use of the rapidly switchable scanning mirror 23,which is less harmful for the sample.

The action of the cylindrical optical element 27 in combination with therapidly switchable scanning mirror 23 is shown again and in detail inFIG. 4, although here, the various elements of the illumination deviceare illustrated only roughly by a few representatives. It is obvioushere that, if a cylindrical optical element 27 is used alone, given theposition of the illumination zoom element 21 sketched here, only part ofthe image field can be covered, so that the fast scanning mirror 23 hasto be used to achieve complete coverage. Without a deflection of thefast scanning mirror 23, the light is imaged into the sample region Palong the beam segment 31, whose outlines are shown here. With adeflection different from zero, the light can be directed into thesample region P along the beam section 32. By means of the additionalscanning movement of the scanning mirror 23, then, a homogeneousillumination of the sample region P can be achieved. If one refrainsfrom using the scanning mirror 23, though, a very fast, stroboscope-likeillumination of the selected sample region becomes possible, whichotherwise would be prevented by the slower movement of the scanningmirror 23.

Arranged additionally in the illumination beam path of the illuminationdevice 19 in FIG. 2 is a second astigmatically acting optical element,which is here, for example, a second cylindrical optical element 33.Instead of a cylindrical optical element 33 with at least onecylindrical lens, astigmatically acting elements with Powell lenses canbe used as well. This second cylindrical optical element is an optionalone, too. It is intended for imaging the fast scanning mirror 23 onto apupil plane and for imaging the angle scanning mirror 28 onto anillumination focal plane. Without this additional second cylindricaloptical element 33 (a correction element), the fast scanning mirror 23and a switching mirror 34 possibly arranged in the same or an equivalentplace in the illumination beam path, intended for light sheet adjustmentand/or deflection of the illumination light into a second illuminationbeam path for illumination from two sides, will only approximately bepositioned in the pupil plane of the illumination optical system. Theconsequence of this is that a position movement in the sample region P,produced by one of the two mirrors 23 or 34, is superposed by a certainshare of an angular movement. This can have an unfavorable effectespecially for the deflecting mirror 34 (which may perhaps also be usedfor adjusting the light sheet in the detection direction), becausedepending on the position of the mirror, an oblique position of thelight sheet relative to the detection plane may result.

However, since the illumination in light sheet microscopy featuresinherent astigmatic properties, these properties can be utilized toachieve exact pupil imaging of both mirrors 23 and 34. The cylindricaloptical element 33 acts on one axis only; it is designed and positionedin such a way that the scanning or deflecting mirror that also acts inthis axis is imaged onto the pupil. Furthermore, the beam path isdesigned so that the mirror which acts in the other axis and on whichthe action of the cylindrical optical element 33 has no influence isalso imaged exactly. A superposition of angular shares on a positionmovement in the sample region P can thus be avoided. Furthermore, thecylindrical optical element 33 can be so designed that the anglescanning mirror 28 is imaged exactly onto the sample plane. Thus, asuperposition of the angular movement with a position movement in thesample region P is avoided. As the two mirrors 23 and 34 are arrangedvery close to each other, the cylindrical optical element 33 isconfigured to have a very long focal length.

FIG. 5 shows a microscope, which may be provided with an illuminationdevice 19, e.g. as illustrated in FIG. 2 and a detection device 1, e.g.as shown in FIG. 1, and in which the illumination device 19 additionallycomprises means for deflecting illumination light into anotherillumination beam path 36 and for generating another light sheet withcorresponding properties, i.e., one that extends in the direction of theillumination axis X and in the direction of the transverse axis Yapproximately planar. In the illumination beam path 35 and in the otherillumination beam path 36, essentially identical optical elements arearranged, and both beam paths have the same optical path lengths; theone light sheet and the other light sheet are aligned with each other insuch a way that they illuminate the sample region P from oppositedirections on the same illumination axis X.

The illumination device 19 further comprises switching means forswitching the illumination light between the illumination beam path 35and the other illumination beam path 36. The switching means comprise arapidly switchable switching element with a switching interval of lessthan 10 ms, with a specified integration time of the array detector 4and the switching interval of the switching element being tuned to eachother in such a way that the sample region P is illuminated at leastonce from each direction on the illumination axis X during theintegration time.

Here, the switching element is designed as a rapidly switchableswitching mirror 34. For example, the switching mirror 34 may begalvanometer-driven. The switching mirror 34 is arranged in a plane thatis conjugate with the illumination pupil plane. In this way, rapidlyswitchable two-beam illumination can be achieved. Depending on theposition of the switching mirror 34, the light is deflected either intothe illumination beam path 35 or—with the aid of a deflecting mirror37—into the other illumination beam path 36. Depending on the positionof the switching mirror 34, thus, the light reaches either theillumination objective 20 or the illumination objective 20′. Both beampaths are designed to have the same optical elements and the sameoptical path lengths.

Instead of a switching mirror 34, other switching elements may be usedas well, such as, e.g., acousto-optical or electro-optical switchingelements, which may operate on a reflective or transmissive basis.

What is important is the short switching interval compared to theintegration time of the array detector, which, as a rule, is not longerthan 20 ms, as otherwise vibrations could have a negative effect; theswitching interval must be so dimensioned that the sample region isilluminated at least once from each direction within the integrationtime. Multiple scans from each direction are also feasible if theswitching interval is small enough, e.g., less than 5 ms.

Although this setup does not permit simultaneous illumination of thesamples from both sides, the fact that the switching mirror 34 has veryshort switching intervals makes it possible to achievequasi-simultaneous illumination if this should be required. For thispurpose, it is only necessary to switch the illumination from oneillumination beam path to the other once within the integration time ofthe camera, so that the sample appears in the image as being illuminatedsimultaneously from both sides, although in fact the illumination issequential.

In effect, then, the sample in the sample region P is illuminatedsimultaneously from opposite directions along the illumination axis X,in that, during the integration time of the array detector 4, theillumination light is switched from one illumination beam path to theother at least once.

FIG. 6 shows, as an example, a sample 38 in which sequentialillumination is not absolutely necessary. FIG. 6 a shows the idealsample image without scatter artefacts. FIG. 6 b shows the real imagesobtained with unilateral light sheet illumination from left (I) andright (II). Here, the sample 38 scatters so much that the two imagesproduced with unilateral illumination do not show any structures incommon; a meaningful generation of a common image is hardly possible, ifat all, in this case. This is also the case if some light-blockingenvironment that is essentially nontransparent exists in the middle ofthe sample. This is the case, at least in part, e.g., with zebra fishembryos, where the embryo's yolk sac blocks the light. Because of thepossible speed gain resulting from faster image recording and theomission of subsequent image processing, simultaneous illumination fromboth sides can make sense here. Although no genuinely simultaneousillumination is possible by means of the fast switching mirror 34described above, what is possible is a quasi-simultaneous recording,which in effect corresponds to simultaneous illumination. During theintegration time of the camera, fast switching-over is performed, whichto the user is virtually imperceptible in the final image.

On the other hand, however, the fact that no genuinely simultaneousillumination of the sample is possible will avoid disadvantagesoccurring in some other samples. Such a sample is shown in FIG. 6 assample 39; it may be a cell cluster, for example. FIG. 6 a shows theideal sample image with sharp structures throughout the sample region.FIG. 6 c shows the real images obtained with unilateral light sheetillumination from left (I) and right (II). Scatter is so pronounced herethat with unilateral illumination, while all parts of the sample remainvisible, a quality loss from left to right or from right to left isevident. Here, simultaneous two-beam illumination is not appropriate, asthe quality loss should be avoided if possible. Sequential illuminationmakes more sense here. However, sequential illumination must beaccomplished very quickly, so that, when a stack of images in thedetection direction Z is recorded, there is the possibility toilluminate each individual plane from both sides. Whereas motor-drivenswitching mirrors have too long switching intervals, with vibrationsbeing possibly transmitted to the mirror, which impairs quality, thefast switching intervals needed, i.e., less than 10 ms, can readily beachieved by means of fast galvanometer-driven mirrors.

The setup shown in FIG. 5 further has the advantage that the switchingmirror 34 is arranged in a pupil plane and, therefore, the positionaloffset produced by the deflection of the switching mirror 34 and thescanning objective 24 is utilized for switching. Of course, other setupsare feasible in which the angular offset is directly utilized forswitching. Another advantage of the setup shown is the possibility touse the switching mirror 34, by means of a slight offset, simultaneouslyfor light sheet adjustment in the detection direction Z.

The illumination device shown in FIG. 5 may also comprise, although notshown, light sheet generation means for generating a rotationallysymmetric light beam—e.g., by means of a scanning mirror 23 and thescanning objective 24—to be used for light-sheet-like scanning of thesample region along the transverse axis, as well as optional anglescanning means—e.g., in the form of an angle scanning mirror 28—, bymeans of which an angle between the light sheet and the illuminationaxis can be varied.

Herein it may be of advantage that the illumination device comprises anastigmatically acting optical element, preferably a cylindrical opticalelement, arranged in the one illumination beam path or in the otherillumination beam path and intended for the correct imaging of theswitching mirror or of the scanning mirror onto a pupil plane. Whereasthe one of the two mirrors is correctly imaged onto the pupil plane, theimaging of the other mirror would not be correct without such anadditional astigmatically acting element; this, then, is an opticalcorrection system for the correct imaging of both mirrors.

An interesting application will be possible if the array detector hasareas of pixels that can be read out separately and for which differentintegration times can be specified, as it is the case, e.g., with modernarray detectors on CMOS basis. Here, the switching interval of theswitching element can additionally be synchronized also with thesedifferent integration times. This can also be combined with theembodiment shown in FIG. 4.

FIG. 7 and FIG. 8 show splitting means for splitting the detection beampath into two branch beam paths 14 and 15, with a spatially resolvingarray detector 4 or 4′, respectively, onto which the light to bedetected is imaged, being arranged in each of the branch beam paths 14and 15, and with the splitting means comprising at least one dichroicbeam splitter 13 or 40, respectively. Both setups can be incorporatedinto a detection device 1 which is shown, e.g., in FIG. 1 and which canbe combined with an illumination device 19 as shown in FIG. 2.

Thanks to the splitting of the detection beam path into two branch beampaths 14 and 15, and with simultaneous suppression of the excitationlight with emission filters and bandpass filters, different spectralranges can be detected via the branch beam paths 14, 15. This makes itpossible to record several color ranges, if, e.g., the sample is labeledwith fluorophores of different colors. If more than two colors are used,sequential recordings can be made in the so-called multitrack mode bychanging the excitation lines and, perhaps, the dichroic beam splitters,bandpass filters or emission filters.

The spatially resolving array detectors 4 and 4′ may be CCDs, EMCCDs,CMOS chips or the like, which may be integrated in the cameras employedby users. For mounting such cameras, it is expedient to provide astandard C-mount interface (not shown) that allows the fitting ofdifferent camera types and models. The C-mount tolerances of laterally0.2 mm and the orientation, i.e., the image rotation of the camera, canbe compensated, e.g., by adjusting screws.

In the embodiment shown in FIG. 7, the dichroic beam splitter 13 isarranged in the beam path in the near-infinity space 12 with referenceto the spatially resolving array detectors 4 and 4′. The dichroic beamsplitter 13 has a thickness of at least 3, preferably of at least 4 mm.In this way, image artefacts, particularly defocusing and astigmatism,which are known in prior art and are caused by surface tensions on thebeam splitter, can be eliminated. Optical imaging elements 5 and 5′,respectively, for imaging the light to be detected onto the respectivearray detector 4, 4′, are arranged in each of the two branch beam paths14 and 15.

As an option, the optical imaging elements 5 and 5′ can, as shown here,be arranged so as to be shiftable in the respective branch beam paths 14and 15, so that focusing on the respective array detector is possible.This can be of advantage, e.g., if a longitudinal chromatic aberrationof the detection optical system has to be corrected. Since what isdetected are individual spectral ranges or wavelengths only, veryprecise adaptation to the wavelength to be detected is possible. If, inaddition, other means for focus shifting are provided, such as, e.g., adetection zoom element 6, it is sufficient to arrange a shiftableimaging element in one of the two branch beam paths, while the elementin the other branch beam path may be fixed.

In at least one of the two branch beam paths 14 and 15, at least onewobble plate for generating a beam offset along two orthogonaldirections transversely to the detection axis Z is arranged in addition.If only one wobble plate is used, it needs to be adjusted in bothorthogonal directions. With equivalent effect, two wobble plates can beused. One of them is then used to adjust the beam offset in onedirection, and the other is used to adjust it in the direction normal toit. FIG. 7 shows, for one of the two beam paths, i.e. the transmissionbeam path 14, two wobble plates 16 and 17 for generating a beam offsetalong two mutually orthogonal directions transversely to the detectionaxis Z.

One can use the wobble plate 16, e.g., for generating a beam offset inX-direction, and wobble plate 17 for generating a beam offset inY-direction. In the other branch beam path 15, the reflection beam path,no wobble plates are required. In this way, an image overlay of the twodetection channels can be achieved, which can also be accomplishedautomatically, provided that a calibration has been performed before bymeans of a calibration objective arranged and selectable in anintermediate image plane of the detection beam path, or by means of asuitable transmission pattern, e.g., a transmission grid pattern 7. Oncethe beam offset has been determined, the images of both array detectors4, 4′ can readily be superposed, and the user is shown only thesuperposition.

Focus adjustment can be achieved not only by means of a shifting of theoptical imaging elements 5, 5′, but also by arranging a wedge assembly42 consisting of two optical wedges that protrude into the beam path andcan be shifted transversely to the beam direction. The wedges are simpleglass wedges that can be moved towards each other or away from eachother. In this way, a variable, additional glass path is moved into thebeam path or out of it, which causes a corresponding shifting of thefocal plane of the optical imaging element 5 or 5′, respectively. Thewedge assembly 42 is arranged between the respective optical imagingelement 5 or 5′ and the respective array detector 4 or 4′ in thedivergent part of the beam path, but it is not shown in FIG. 7.

Another embodiment for two-channel detection is shown in FIG. 8. Here, adichroic beam splitter 40 of any thickness is arranged in the divergentpart of the imaging beam path, i.e. between the optical imaging element5 and the array detectors 4 and 4′. In one of the two branch beam paths,viz. in the branch beam path 14 for the light transmitted by the beamsplitter, a glass plate 42 is arranged between the array detector 4 andthe beam splitter 40, with an angle of approximately 90° between theglass plate and the beam splitter 40. This glass plate 41 has the samethickness as the beam splitter 40; in this way, the astigmatism causedby the beam splitter 40 in the transmission direction is corrected.

In a preferred embodiment, the glass plate 41 can be used simultaneouslyas a wobble plate for adjusting the offset between the two camerachannels. The glass plate 41 is needed only in the transmission beampath, i.e., in the branch beam path 14, which images the lighttransmitted by the beam splitter 40 onto the array detector 4. In thereflected branch beam path 15 in FIG. 8, the above-mentioned wedgeassembly 42 is arranged, with which the focal plane of the opticalimaging element 5 can be shifted by shifting the two glass wedgestowards or away from each other (as indicated by the double-headedarrow). Such a wedge assembly 42 may also be arranged in thetransmission beam path; in this case one can, e.g., drop the shifting ofthe optical imaging element 5 so that this need not be shiftable. Inprinciple, though, it is sufficient to have the optical imaging element5 shiftable and to introduce a wedge assembly 42 into one of the twobeam paths. Whereas then, e.g., as in the example shown in FIG. 8, thefocal plane for the transmission branch beam path is varied only bymeans of the optical imaging element 5, the focal plane for thereflection beam path can be further adjusted by means of the wedgeassembly 42.

It is understood that the features mentioned before are applicable notonly in the combinations stated but also in other combinations or asstand-alone features without leaving the scope of the present invention.The individual elements described above can all be combined with eachother. It is also possible to use individual elements without otherelements shown; for example, one can do without a zoom device when twobranch detection beam paths are used simultaneously. The combination ofillumination from two directions can be combined with detection in twochannels, and this with the illumination and detection zoom elements andsimultaneous internal focusing. Altogether, the arrangements describedabove exhibit substantial improvements over the arrangements known inprior art.

LIST OF REFERENCE NUMBERS

-   1 detection device-   2 detection objective-   3 tube lens unit-   4, 4′ array detector-   5, 5′ imaging element-   6 detection zoom element-   7 transmission grid pattern-   8 tube lens-   9, 10 deflecting mirror-   11 beam coupler-   12 near-infinity space-   13 beam splitter-   14, 15 branch beam path-   16, 17 wobble plate-   18 emission filter-   19 illumination device-   20 illumination objective-   21 illumination zoom element-   22 telescope changer-   23 scanning mirror-   24 scanning objective-   25 tube lens element-   26 mirror-   27 cylindrical optical element-   28 angle scanning mirror-   29, 30 beam path-   31, 32 beam segments-   33 cylindrical optical element-   34 switching mirror-   35 illumination beam path-   36 other illumination beam path-   37 deflecting mirror-   38, 39 sample-   40 beam splitter-   41 wedge assembly-   42 glass plate

What is claimed is:
 1. A microscope, comprising: an illumination device, that generates a sheet of light extending along an illumination beam path for illuminating a sample region, the sheet of light having an illumination axis X in the direction of the illumination beam path and a transverse axis Y lying across the illumination axis X, the sheet of light having an approximately planar extension; and a detection device used to detect light that is radiated by the sample region along a detection beam path in a direction of a detection axis Z, wherein the angle between the illumination axis X and the detection axis Z are unequal to zero and the angle between the transverse axis Y and the detection axis Z are unequal to zero, wherein the detection device comprises a detection objective having a front lens, the detection objective positioned in the detection beam path, and the detection device further comprises an optical detection element located separately from the front lens of the detection objective, the optical detection element adjustable independently of said front lens of the detection objective, whereby adjustment of the optical detection element provides at least one of: a) continuously and variably changing the size of a detection image field, and b) continuously shifting a detection focal plane in the sample region wherein the illumination device comprises at least one illumination objective and an optical illumination element, the illumination objective comprising a front lens, the optical illumination element positioned separately from the front lens of the illumination objective, the optical illumination element being adjustable independently from the front lens, whereby the extension of the sheet of light in the direction of the detection axis Z is continuously variable by adjustment of the optical illumination element; wherein the optical illumination element is configured as a illumination zoom element: wherein the optical detection element is configured as a detection zoom element, and wherein the illumination zoom element and the detection zoom element are coupled via a control circuitry for setting the illumination zoom element as a function of the size of the detection image field provided by the detection zoom element.
 2. The microscope as claimed in claim 1, wherein the detection device is provided with a tube lens unit which is located separately from the detection element and from the detection objective, and the tube lens unit is configured to shift the detection focal plane in the sample region.
 3. The microscope as claimed in claim 1, wherein the optical illumination element that is configured as a illumination zoom element comprises at least three lens components which are adjustable independently of each other.
 4. The microscope as claimed in claim 1, further comprising a transmission grid element moveable from a first position to a second position and adapted to collect an intermediate image transmission pattern sample, wherein the intermediate image transmission pattern sample is collected when the transmission grid is disposed in the first position in an intermediate image plane of the detection beam path.
 5. A microscope, comprising: an illumination device, that generates a sheet of light extending along an illumination beam path for illuminating a sample region, the sheet of light having an illumination axis X in the direction of the illumination beam path and a transverse axis Y lying across the illumination axis X, the sheet of light having an approximately planar extension; and a detection device used to detect light that is radiated by the sample region along a detection beam path in a direction of a detection axis Z, wherein the angle between the illumination axis X and the detection axis Z are unequal to zero and the angle between the transverse axis Y and the detection axis Z are unequal to zero, wherein the detection device comprises a detection objective having a front lens, the detection objective positioned in the detection beam path, and the detection device further comprises an optical detection element located separately from the front lens of the detection objective, the optical detection element being adjustable independently of said front lens of the detection objective, whereby adjustment of the optical detection element provides at least one of: a) continuously and variably changing the size of a detection image field, and b) continuously shifting a detection focal plane in the sample region; wherein the illumination device comprises at least one illumination objective and an optical illumination element, the illumination objective comprising a front lens, the optical illumination element positioned separately from the front lens of the illumination objective and the optical illumination element being adjustable independently from the front lens of the illumination objective, whereby an illumination focal plane in the sample region is continuously adjustable by adjustment of the optical illumination element; wherein the optical illumination element is configured as a illumination zoom element; and wherein the optical detection element is configured as a detection zoom element, and wherein the illumination zoom element and detection zoom element are coupled via a control circuitry for setting the illumination zoom element as a function of the size of the detection image field provided by the detection zoom element.
 6. The microscope as claimed in claim 5, wherein the optical illumination element that is configured as a illumination zoom element comprises at least three lens components which are adjustable independently of each other.
 7. The microscope as claimed in claim 5, wherein the detection device is provided with a tube lens unit which is located separately from the detection element and from the detection objective, and the tube lens unit is configured to shift the detection focal plane in the sample region.
 8. The microscope as claimed in claim 5, further comprising a transmission grid element moveable from a first position to a second position and adapted to collect an intermediate image transmission pattern sample, wherein the intermediate image transmission pattern sample is collected when the transmission grid is disposed in the first position in an intermediate image plane of the detection beam path.
 9. A method, comprising: adjusting an illumination device for a selective plane illumination microscopy microscope, wherein said illumination device generates a substantially planar light sheet extending along an illumination beam path for illuminating a sample region, the sheet of light having an illumination axis X_(ill) along the illumination beam path and a transverse axis Y_(trans) lying across the X_(i11), the sheet of light having an approximately planar extension; and detecting via a detection device the light sheet along a detection beam path that defines a detection axis Z_(dx), wherein the angle between the X_(ill) and the Z_(dx) are unequal to zero and the angle between the Y_(trans) and the Z_(dx) are unequal to zero, wherein the detection device comprising a detection objective having a front lens, the detection objective positioned in the detection beam path, the detection device further comprises an optical detection element which is located separately from the front lens of the detection objective, the optical detection element being adjustable independently of said front lens of the detection objective, whereby adjustment of the optical detection element provides at least one of: a) continuously and variably changing the size of a detection image field, and b) continuously adjusting a detection focal plane in the sample region; and wherein the detecting step includes collecting a plurality of multiple track micrograph images with at least some of said plurality collected images collected at different wavelengths of light sheet radiation along the detection beam path.
 10. The method according to claim 9, wherein the detecting device comprises one of a charge-coupled device, a digital camera, and a complementary metal oxide semiconductor chip.
 11. The method according to claim 9, further comprising splitting the illumination beam path so that a pair of beam paths are formed and each of the pair of beam paths impinge upon the sample region from opposing sides of the sample region.
 12. The method according to claim 9, further comprising: moving a transmission grid element from a first position to a second position and to collect an intermediate image transmission pattern sample at the first position, wherein the intermediate image transmission pattern sample is collected in an intermediate image plane of the detection beam path.
 13. The method according to claim 9, further comprising: measuring a temperature of a fluid residing in a sealed container that envelops the sample region; and with reference to a refraction index of the fluid residing in the sealed container, adjusting at least one of: a detection focal plane and the temperature of the fluid.
 14. The method according to claim 9, wherein the detecting step includes collecting a plurality of multiview image stack images with at least some of said plurality of collected images collected at differing sample angles of the light sheet traveling along the detection beam path. 